Roles of the pro-apoptotic factors CaNma111 and CaYbh3 in apoptosis and virulence of Candida albicans

Candida albicans, a commensal and opportunistic pathogen, undergoes apoptosis in response to various stimuli, including hydrogen peroxide, acetic acid, and antifungal agents. Apoptotic processes are highly conserved among mammals, plants, and fungi, but little is known about the apoptosis-regulating factors in C. albicans. In this study, C. albicans homologs of the putative apoptosis factors were identified by database screening followed by overexpression analysis. CaNma111, a homolog of the pro-apoptotic mammalian HtrA2/Omi, and CaYbh3, a homolog of BH3-only protein, yielded increased apoptotic phenotypes upon overexpression. We showed that CaNma111 and CaYbh3 functions as pro-apoptotic regulators by examining intracellular ROS accumulation, DNA end breaks (TUNEL assay), and cell survival in Canma111/Canma111 and Caybh3/Caybh3 deletion strains. We found that the protein level of CaBir1, an inhibitor-of-apoptosis (IAP) protein, was down-regulated by CaNma111. Interestingly, the Canma111/Canma111 and Caybh3/Caybh3 deletion strains showed hyperfilamentation phenotypes and increased virulence in a mouse infection model. Together, our results suggest that CaNma111 and CaYbh3 play key regulatory roles in the apoptosis and virulence of C. albicans.


Results
Overexpression of putative apoptosis factors in C. albicans. To investigate putative apoptosisregulating factors in C. albicans, we constructed overexpression strains of five genes: CaBIR1, CaNMA111, CaYBH3, CaDHH1 and CaPAT1 (Fig. 1). These genes were identified from the Candida Genome Database based on their amino acid sequence similarities with homologous proteins in S. cerevisiae and mammals. CaBir1, a single IAP in C. albicans, was shown to inhibit apoptosis by reducing caspase-like activity under an oxidative stress condition 19 . CaNma111 (nuclear mediator of apoptosis) is a homolog of the pro-apoptotic serine protease, HtrA2/Omi. In mammals and S. cerevisiae, HtrA2/Omi regulates apoptosis by binding and degrading cellular IAPs 15,16 . Ybh3 is the yeast homolog of the BH3-only protein, which contains a BCL-2 homology domain (BH3) 13,14 . We identified its homolog, CaYbh3, in C. albicans. Dhh1 and Pat1, which are known as the components of P-bodies (processing bodies, mRNA granules) in S. cerevisiae, function as mRNA-decapping activators 20,21 . CaDhh1, which was identified in a previous work, was shown to be localized to P-bodies in C. albicans 22 . CaPat1 was identified as C. albicans homolog in this study. For ectopic overexpression, each target gene was cloned downstream of the ACT1 promoter in plasmid pPR671, and the constructed plasmid was chromosome-integrated into the wild-type C. albicans strain 23 . Overexpression of CaNma111 or CaYbh3 yields increased apoptotic phenotypes. Apoptosis is characterized by several morphological and biochemical features including chromatin condensation, accumulation of reactive oxygen species (ROS), and increased caspase activity 6,24 . In each overexpression strain, we determined the intracellular amount of ROS by staining cells with the fluorescent dye, H 2 DCFDA (Fig. 2). Compared to wild-type BWP17 cells, overexpression strains OECaNMA111 and OECaYBH3 showed increased frequencies of H 2 DCFDA-positive cells before and after apoptosis-inducing H 2 O 2 treatment. OECaBIR1 and OECaPAT1 showed decreased ROS accumulation compared to the wild-type. OECaDHH1 showed a ROS level higher than that of wild-type cells prior to H 2 O 2 treatment but similar to that of wild-type cells after H 2 O 2 treatment.
Next, we measured the caspase-like activity in the overexpression strains using the caspase substrate, D 2 R (Asp 2 Rhodamine 110). Cells were stained with D 2 R and the frequency of fluorescent D 2 R-positive cells was calculated (Fig. 3). In the wild-type strain, BWP17, few cells were fluorescent in H 2 O 2 -untreated cells, but numerous fluorescent cells were observed after 30 min of H 2 O 2 stress. OECaNMA111 and OECaYBH3 showed increases in the frequency of fluorescent cells compared to the wild-type strain with or without oxidative stress. In contrast, OECaBIR1 and OECaPAT1 showed little fluorescence, regardless of H 2 O 2 treatment. OECaDHH1 showed an increase in the number of fluorescent cells before H 2 O 2 treatment, but only a slight increase after H 2 O 2 treatment. These patterns of caspase-like activity in each overexpression strain were closely associated with the ROS accumulation level.
Deletion of CaNMA111 or CaYBH3 decreased apoptotic phenotypes. To further investigate the roles of CaNMA111 and CaYBH3 in apoptosis, we sequentially deleted the two copies of CaNMA111 or CaYBH3 to construct the Canma111/Canma111 and Caybh3/Caybh3 deletion strains, respectively. The wildtype and deletion mutant strains were compared for apoptotic hallmarks, including ROS accumulation, nuclear  CaNma111 downregulates the apoptosis inhibitor, CaBir1. The mammalian serine protease, Omi/ HtrA2, promotes apoptosis by binding and degrading IAP family proteins 25,26 . Consistently, the yeast IAP, Bir1, was shown to be a substrate for Nma111 in S. cerevisiae 15 . We repeatedly observed a very faint protein band when we assessed chromosome-tagged CaBir1-GFP or CaBir1-myc in a wild-type background (data not shown). To ask whether CaNma111 is one of the proteases responsible for the degradation of CaBir1, we compared CaBir1myc levels in wild-type and Canma111/Canma111 cells. Here, CaBir1-myc was expressed under the control of the ACT1 promoter of the pPR671 vector. We observed an increased level of CaBir1-myc in Canma111/

Deletion of CaNMA111 or significantly increases filamentous growth and virulence. C. albi-
cans is an opportunistic pathogen and switches rapidly among the budding yeast, pseudohyphal, and hyphal forms in response to environmental changes 27,28 . This morphogenetic switching is particularly associated with virulence. In addition, it has been suggested that the morphological state affects apoptotic cell death 29 . We therefore examined whether the pro-apoptotic regulators, CaNma111 and CaYbh3, are involved in the filamentous growth or virulence of C. albicans. Colony morphologies of the wild-type, Canma111/Canma111, and Caybh3/Caybh3 strains were examined on hyphae-inducing solid medium. As shown in Fig. 5A, Canma111/ Canma111 and Caybh3/Caybh3 mutant cells exhibited hyperfilamentation phenotypes on solid Spider medium, compared with wild-type cells. Interestingly, the mutant strains showed filamentous growth on YEPD complete   www.nature.com/scientificreports/ medium (data not shown). The hyperfilamentation phenotypes of Canma111/Canma111 and Caybh3/Caybh3 mutant strains were also evident in liquid medium supplemented with 10% serum (Fig. 5B). The virulence of the Canma111/Canma111 and Caybh3/Caybh3 strains was tested in a tail vein-infection model with BALB/c mice. Two groups of mice (n = 10) were challenged with wild-type and mutant cells and survival was monitored for up to 30 days. Mice infected with Canma111/Canma111 or Caybh3/Caybh3 mutant strain showed more rapid weight loss and decreased survival than those infected with wild-type BWP17 (Fig. 5C). These results demonstrate that CaNMA111 and CaYBH3 play important roles in the virulence of C. albicans.

Discussion
We analyzed five candidate regulators for apoptosis in C. albicans and found that overexpression of CaNma111 or CaYbh3 yielded pro-apoptotic features, while that of CaBir1 or CaPat1 yielded anti-apoptotic feature. CaNma111 and CaYbh3 were further characterized by constructing the deletion mutant strains. Gene overexpression mimics gain-of-function mutations, and thus offers a useful approach for revealing pathways or pathway components in the diploid pathogen, C. albicans [30][31][32] . The overexpression phenotypes of the apoptosis inhibitor, CaBir1, were consistent with our recent report that the Cabir1/Cabir1 mutant strain showed increased apoptotic phenotypes, such as ROS accumulation and DNA fragmentation, under apoptosis-inducing conditions 19 . We analyzed CaDhh1 and CaPat1 because our previous work showed that CaEdc3, another component of P-bodies, is involved in apoptosis 33 . CaEdc3 contributes to the expression of CaMca1 expression and thereby functions as a pro-apoptotic factor. In our overexpression analysis, CaPat1 was suggested to be anti-apoptotic factor. We suggest that CaDhh1, CaEdc3, and CaPat1 could all participate in apoptosis, with each playing a distinct role. CaDhh1 and CaPat1 show protein interactions with each other but differ in their functional domains, intracellular locations, and mRNA targets 20,34 .
Here, we report our results from the deletion mutant analysis of CaNMA111 and CaYBH3. The decreased apoptotic phenotypes of Canma111/Canma111 and Caybh3/Caybh3 mutant cells suggest that CaNma111 and CaYbh3 function as pro-apoptotic regulators in C. albicans. HtrA2/Omi, which is a mammalian counterpart of CaNma111, has been identified as a direct IAP-binding protein 16,26 . It exerts pro-apoptotic character effects, possibly by disruption of the IAP-caspase interaction. Studies have shown that the serine protease, HtrA2/Omi,

Survival (%)
Days aŌer infecƟon www.nature.com/scientificreports/ can degrade mammalian IAP and XIAP 25,26 . We repeatedly observed very faint protein band corresponding to CaBir1-GFP or CaBir1-myc in a wild-type background (data not shown). We speculated that the full-length CaBir1 protein could be a target of proteolytic degradation. Our observation that the CaBir1-myc protein level was increased in Canma111/Canma111 cells may support this notion. However, future work is needed to assess whether CaNma111 could be among the proteases responsible for CaBir1 degradation. The hyperfilamentation phenotypes and increased virulence of the Canma111/Canma111 and Caybh3/ Caybh3 mutant strains were particularly interesting, as these findings suggest that the pro-apoptotic regulators, CaNma111 and CaYbh3, exert repressive actions on filamentation and pathogenicity in C. albicans. It remains unknown whether the pro-apoptotic roles of CaNma111 and CaYbh3 overlap with their functions during morphogenesis. One possible explanation is that CaNma111, which is a serine protease responsible for degrading the apoptosis inhibitor, CaBir1, could be involved in the processing or breakdown of regulatory factors crucial for filamentous growth. Further studies will be needed to uncover the downstream targets of CaNma111 or CaYbh3 protease activity during morphogenesis. Regarding CaYbh3, we speculate that a putative BH3 domain within this C. albicans protein could be responsible for mitochondria-driven ROS accumulation and/or the release of apoptotic factors 13 . During hyphal morphogenesis, C. albicans produces a burst of ROS that is mainly located at the hyphal tip 35,36 . Further investigation is needed to examine whether the repressive function of CaYbh3 during filamentous growth could also be associated with changes in the ROS level.
It is noteworthy that the pro-apoptotic regulator, CaMca1 metacaspase, was shown to be required for filamentation and pathogenicity 37 . Cells harboring the apoptosis-defective deletion of CaMCA1 or the catalytic-site mutation CaMCA1 c292 , showed defects in filamentation and virulence. It has been suggested that S. cerevisiae metacaspases, which are responsible for apoptosis, are also involved in nonapoptotic characteristics and processes, such as longevity, the fitness of growing cells, and protein clearance 38,39 . We speculate that the downstream targets of CaNma111 protease and CaMca1 metacaspase could act to either promote or repress filamentous growth and other nonapoptotic processes.
Various regulatory elements involved in the yeast-to-hyphal transition has been identified in C. albicans 27,40,41 . The Ras-cAMP-PKA and the MAPK pathway operate to promote the yeast-to-hyphal transition and the transcription factors, such as Cph1 and Efg1, are targets of these pathway responses in C. albicans. Activation of Ras-signaling was shown to accelerate apoptotic responses under treatment with acetic acid or H 2 O 2 42 . However, little is known about the interrelationship between cell death and morphogenesis. The quorum-sensing molecule, farnesol, inhibits the yeast-to-hyphal switch, but this triggers apoptosis 4,5 . Going forward, additional work is needed to improve our understanding of the detailed regulatory points and components involved in the apoptotic responses and pathogenicity of C. albicans.

Materials and methods
Strains, plasmids, and culture conditions. The C. albicans strains and plasmids used in this study are listed in Table S1. Constructions of the Canma111/Canma111 and Caybh3/Caybh3 deletion strains were essentially as described previously 22,40 . We used plasmids pJI434 and pJI435 for CaNMA111 deletion, and plasmids pJI436 and pJI437 for CaYBH3 deletion. These plasmids carried the deletion cassettes, hph-URA3-hph and hisG-URA3-hisG, respectively. Each disruption was verified by PCR. Overexpression strains were constructed using the pPR671-derived plasmids, pJI426-pJI432. Each target gene was amplified using a primer set (Table S2), and the PCR fragment was digested with MluI/XmaI and ligated into the MluI and XmaI sites of pPR671 22 . Each pPR671-derived plasmid was linearized by StuI and transformed into the wild-type BWP17 strain. Chromosome integrations were verified by PCR and protein expressions were analyzed by Western blot.

ROS accumulation assay.
Western blot analysis. Total protein preparation and Western blotting were performed as previously described 22 . Myc-tagged proteins were detected with anti-myc antibody (Roche, USA). HRP-conjugated antimouse IgG antibody (Santa Cruz Biotechnology, USA) was used as the secondary antibody. Tubulin protein was used as a loading control, and was detected with a monoclonal anti-α-tubulin antibody (Sigma-Aldrich, USA). Protein bands were visualized using an Enhanced Peroxidase Detection (EPD) Western reagent kit (Elpis-Biotech, KR).
Assessment of virulence in a murine infection model. Cells were grown overnight in SC-Ura medium and washed twice with sterile physiological saline. Seven-week-old female BALB/c mice were infected via lateral tail vein injection with 6 × 10 5 CFU (colony forming unit) in a 100-μl volume. Ten mice were inoculated per test strain, and host survival was monitored over 30 days. All animal experiments were approved by the Animal Experiment Ethics Committee of Chungnam National University (approval No. 202006A-CNU-120, July 2020) and performed in accordance with the guidelines of the Ethics Training Guidelines for Experiments on Animals of CNU Animal Research Center. This study additionally adheres to standards articulated in the ARRIVE guidelines.